Method of manufacturing micro chamber plate with built-in sample and analytic micro chamber plate, analytic micro chamber plate and apparatus set for manufacturing analytic micro chamber plate with built-in sample

ABSTRACT

A method of manufacturing a micro-chamber plate with a built-in sample, including: settling a micro-chamber plate for sample injection at a micro-chamber plate receiving part formed with an upper opening; disposing a cover for micro-chamber plate receiving part to cover the upper opening, the cover for micro-chamber plate receiving part having a provisional storing part and an auxiliary covering part connected with the provisional storing part and formed with a through-hole for auxiliary covering part; and manufacturing a micro-chamber plate with a built-in sample by putting the micro-chamber plate receiving part, on which the cover is disposed, into a centrifugal separator which can apply vacuum, applying centrifugal force and injecting a sample solution provisionally stored in the provisional storing part into the micro-chamber plate through a vessel communication part which is formed at the provisional storing part to be communicated with the micro-chamber plate receiving part.

CROSS REFERENCE TO PRIOR APPLICATION

This application is Divisional Application of U.S. patent applicationSer. No. 13/810,534 filed on Jan. 16, 2013 now U.S. Pat. No. 9,151,714,which is a National Stage Patent Application of PCT International PatentApplication No. PCT/KR2011/004010 filed on Jun. 1, 2011 under 35 U.S.C.§ 371, which claims priority to Korean Patent Application No.10-2010-0071651 filed on Jul. 23, 2010, which are all herebyincorporated by reference in their entirety.

BACKGROUND

The present invention relates to micro-chamber plate, and particularlyto an analytic micro-chamber plate in which multiple reaction solutionscontaining a primer or a probe selectively reacted with each nucleicacid are reacted without cross contamination in order to analyze abiological sample solution containing multiple nucleic acids, therebymeasuring and analyzing a fluorescence value in real time.

Further, the present invention related to a method of manufacturing theanalytic micro-chamber plate.

Further, the present invention relates to a method of manufacturing amicro-chamber plate with a built-in sample, which is used in themanufacturing of the analytic micro-chamber plate.

Further, the present invention relates to an apparatus set formanufacturing the sample containing micro-chamber plate.

Generally, a micro-chamber is a container which is formed of siliconwafer, glass, metal, plastic or the like and in which a fine reactionless than a few micro-liters. The micro-chamber plate is a plate inwhich the micro-chambers are arranged in two dimensions and of which oneside surface is formed to be sealed after a sample is injectedtherethrough.

Meanwhile, there has been developed a real-time PCR (Polymerase ChainReaction) method which can measure a fluorescence value increasing inproportion to an amount of genes in real-time, while performing a PCR.

In the real-time PCR method, while the PCR is carried out, thefluorescence value generated from a product of the PCR is measured inevery cycle, and the cycle when the fluorescence value larger than adesired value is generated is checked, thereby quantitatively analyzingan initial concentration of a specific gene in a sample.

In the real-time PCR method, there are some advantages in that anelectrophoresis process following the PCR is not needed, and it ispossible to decide a concentration of a gene having a specific basesequence in the range of 10⁹ or more (“A-Z of Quantitative PCR” editedby Stephen A. Bustin 2004-2006 International University, “Real-time PCR”edited by M. Tevfik Dorak 2006 Taylor & Francis Group).

There had been proposed various kinds of real-time PCR apparatuses forperforming the real-time PCR method. For example, there is aconventional real-time PCR apparatus which can analyze 96 or 384 genesusing a standard 96-well or 394-well plate, thereby analyzing aplurality of samples (Light cycler 480 manufactured by Roche, ABI 7500,7900).

In the conventional real-time PCR apparatus manufactured by Roche, inwhich a reaction sample of 10˜500 μl is used, however, there is aproblem that it is not possible to analyze a large number of genescompared to a large amount of used sample.

In order to solve the problem, various methods which can simultaneouslyanalyze multiple samples in a shorter time by reducing a used amount ofthe reaction sample using a MEMS (Micro Electro Mechanical Systems)technology have been proposed, and thus a method using a micro-chamberarray plate has been also proposed.

The method using a micro-chamber array plate includes a step ofinjecting a reaction solution into a micro-chamber, a step of sealingthe reaction solution in each micro-chamber, and a reacting andanalyzing step. In a method of separately applying a sample solution ineach micro-chamber, a transparent micro-chamber plate for cell cultureis covered by a semi-permeable membrane so as to individually isolatethe micro-chambers, and one cell is cultivated in each micro-chamber,and a Taqman reaction solution is supplied after removing a culturemedium, and the micro-chamber is sealed by transparent oil, and then afluorescence value is measured at a bottom surface of the plate (YASUDA,Kenji EP 1,541,678 A1, JP 2002245900 NUCLEIC ACID ANALYSIS CHIP ANDNUCLEIC ACID ANALYZER).

In the above-mentioned method, however, since different solutions haveto be applied to each micro-chamber using a pipette, much time is spenton that. Particularly, an auto-pipetting system is needed in order toinject the sample in 1,536 or more micro-chambers. Herein, in order toapply the different solutions, it takes lots of time due to a cleaningprocess which has to be performed before applying each of the differentsolutions. Thus, there is a problem in that it is difficult to use the384 plates or more.

Secondly, in order to solve the problem, there had been proposed areactor by E. Tamiya, Hidenori Nagai et al., in which a micro-chamber isformed by treating a silicon wafer in a photolithography process and achemical etching process (Anal. Chem. 2001 73, 1043-1047, Development ofa Micro-chamber Array for Picoliter PCR).

In the reactor, a micro-slide cover glass is used to prevent theevaporation of a PCR solution. However, since cross contamination of thePCR solution is occurred when covering or separating the cover glass, itis inconvenient that the cover glass has to be removed while awater-repellent film is interposed between the cover glass and thewafer, the water-repellent film has to be removed after drying the PCRsolution and then an analysis process has to be performed. Further,there is a problem in that it cannot be used in qPCR technology.

Thirdly, in order to solve the problem of using in the qPCR technology,there had been developed another micro-chamber array by Y. Matsubara etal. belonging to the same laboratory, in which a primer is applied to amicro-chamber formed on a wafer using a micro-array device and thendried (7^(th) International Conference on Miniaturized Chemical andBiochemical Analysis Systems Oct. 5-9, 2003, Squaw Valley Calif. USA).

The micro-chamber array uses a method in which mineral oil is applied ona chip so as to completely cover the micro-chambers, and then a PCRsolution is dripped on the mineral oil of the reactor using a nano-jetpipetting system.

In the method, 1,248 micro-chamber array chips having a volume of 50nano-liters (0.65×0.65×0.2 mm) are manufactured by treating a 1 inch×3inch silicon wafer in a photolithography process and a chemical etchingprocess, a primer and a Taqman probe solution are dripped in themicro-chambers using the nano jet pipetting system and dried, and thenthe mineral oil is coated thereon so that each micro-chamber is isolatedand sealed.

In case of the micro-chamber array manufactured by the third method,since a mixed solution of a Taq DNA polymerase and a sample DNA isinjected on the mineral oil using the nano jet pipetting system so as tobe dripped in each micro-chamber, there is an advantage in that it ispossible to successfully carry out the PCR in the micro-chambers withoutcross contamination of each reaction component.

However, in this method, there are some problems that a separate nanojet pipetting system for micro-array is needed to inject the solution,it takes lots of time to perform the pipetting operation and there isalso a high risk of the cross contamination among the reaction solutionsdue to flowing of the mineral oil when the plate is moved. Further, in atemperature cycling reaction, bubbles are generated at high temperature.Meanwhile, the aqueous solution in each micro-chamber is formed into aglobular shape due to a hydrophobic effect between the oil and theaqueous solution, thereby causing a lens effect. Thus since excitationlight and luminescence is scattered and dispersed upon the opticalmeasurement, the measurement error is increased.

Fourthly, there had been also developed a picotiter plate in whichmicro-chambers are formed by the photolithography process and thechemical etching process like in the third method but a lot morereactions than in the third method can be performed (John H. Leamon etal., A massively parallel PocoTiterPlate based platform for discretepico-liter-scale polymerase chanin reactions, Electrophoresis 2003, 24,3769-3777).

In the fourth method, it is possible to independently carry out 300,000PCRs with an amount of 39.5 pl. However, since a carrier in whichprimers/probes are immobilized is needed, it cannot be applied to areal-time quantitative PCR method in which uniform opticalcharacteristics are required.

Fifthly, in U.S. Pat. No. 5,948,637, there has been proposed a reactorcalled “a film reactor (or a DNA card)” for reacting a small amount ofsample.

The film reactor is form of a three-layered very thin film. Detailedly,a lower film forms a lower surface of the reactor, a middle film forms aside surface of the reactor and an upper film forms an injection hole.After a small amount of sample solution is injected into the filmreactor by using a pipette, the injection hole has to be completelysealed. If the injection hole is not completely sealed, there is aproblem that the reaction solution is evaporated upon the PCR. Further,since the film reactor has a complicated structure in order to treat afew thousands of samples, it is substantially impossible to manufactureit.

Sixthly, in WO 02/40158 and U.S. Pat. No. 6,232,114, there is discloseda reaction plate which can carry out 1,535 fluorescence analysisreactions with a standard ELISA plate scale.

In the sixth method, multiple through-holes are formed in the plate, anda transparent film having a small fluorescence amount is fused so as toform a plurality of reaction vessels. After the sample is received ineach reaction vessel, the reaction vessels are sealed with thetransparent film and the reaction is carried out. Upper and lowersurfaces of the reaction plate are formed to be transparent, andexcitation light is applied through one side surface, and then thefluorescence is measured through the other side surface.

In the sixth method, however, different primer and probe have to berespectively injected into each micro-chamber in order to analyze agreat number of genes. In case of a plate for analyzing a great numberof samples, since a few thousands of different solutions are injected atthe micro-chambers, a special pipetting system such as a nano-literpipetting system is needed, much time is spent on that and alsoerroneous injections may be occurred. Further, since the micro-chambercannot be completely filled with the solution, bubbles are generated,and the water vapors are formed at an upper portion of the micro-chamberwhen raising the temperature, and thus the optical measurement isdisturbed by the scattering.

Seventhly, in PCT/KR2008/005635 invented by the invention of the presentapplication, there is disclosed a reaction plate using a micro-chamberplate that a porous membrane for injecting a sample is formed at oneside surface thereof and an optical measuring part is formed at theother side surface thereof.

In the seventh method, multiple through-holes are formed in the plate,and a transparent film having a small fluorescence amount is fused atone side surface thereof so as to form a plurality of reaction vessels.After the sample is received in each reaction vessel, the other sidesurface thereof is sealed with the porous membrane through which thesample solution can be injected, and the reaction is carried out. In thereaction plate, the sample solution is injected through the porousmembrane, and mineral oil is sealingly dripped on the injection surface,and then excitation light is applied and the fluorescence is measuredthrough the optical measuring part formed at the other side surfacethereof.

However, in the seventh method, since the injection part and the opticalmeasuring part are formed separately, it has a complicated structure.And the oil layer formed on the injection part becomes transparent, andthus a deviation problem in the measurement results may be occurredaccording to a stained state of a bottom surface. Further, the injectionpart on which the mineral oil is dripped may be directed downward inorder to perform the reaction and measurement. At this time, the mineraloil having a relatively lower density than the sample may be introducedinto the micro-chamber, and thus the scattering may be occurred.

Eighthly, in PCT/KR2008/005635, there is disclosed “The micro-chamberplate, manufacturing method thereof”.

However, since the eighth method has a structure that a sample to beinjected is directly applied to a porous membrane, it has some problemsas follows: 1) in case that the injection of the sample is achieved by avacuum, centrifugal force is applied in order to prevent running-out ofthe sample while the vacuum is applied. Herein, discharging of gasthrough pores of the porous membrane is disturbed by the centrifugalforce and surface tension of the sample; 2) since the gas in themicro-chamber is compressed by the centrifugal force and thus a volumethereof is contracted, the gas does not obtain enough buoyancy to getout of the micro-chamber through the membrane, but is remained in theform of small bubbles and then expanded again in the measurementcondition of atmospheric pressure, thereby disturbing the measurement.

Therefore, a new micro-chamber plate is needed, in which the sample canbe easily injected into the plurality of micro-chambers, thecross-contamination is not occurred, light generated from the sample canbe precisely measured in real time without possibility that the opticalmeasuring part is contaminated with the sample or the like.

SUMMARY

An object of the present invention is to provide a micro-chamber plateand a manufacturing method thereof, in which it is prevented that asolution is evaporated in a plurality of micro-chambers needed in areal-time PCR, a fixed temperature enzyme reaction and an LCR (LigaseChain Reaction), it is possible to facilely inject the solution and thusremarkably reduce time required in an injection process, it is preventedthat the solutions in the micro-chambers are mixed with each other, theinjection part and the optical measuring part are integrally formed soas to provide a simple structure, such that fine bubbles are notgenerated, and thus it is possible to more precisely measure thefluorescence value, thereby increasing analyzing accuracy.

To achieve the object of the present invention, the present inventionprovides a method of manufacturing a micro-chamber plate with a built-insample, comprising a step S20 of settling a micro-chamber plate 100 forsample injection at a micro-chamber plate receiving part 200 formed withan upper opening; a step S30 of disposing a cover 310 for micro-chamberplate receiving part so as to cover the upper opening of themicro-chamber plate receiving part 200, the cover 310 for micro-chamberplate receiving part comprising a provisional storing part 312 and anauxiliary covering part 314 connected with the provisional storing part312 and formed with a through-hole 314-1 for auxiliary covering part;and a step S40 of manufacturing a micro-chamber plate 100A with abuilt-in sample by putting the micro-chamber plate receiving part 200,on which the cover 310 for micro-chamber plate receiving part isdisposed, into a centrifugal separator which can apply vacuum, applyingcentrifugal force and injecting a sample solution provisionally storedin the provisional storing part 312 into the micro-chamber plate 100 forsample injection through a vessel communication part which is formed atthe provisional storing part 312 so as to be communicated with themicro-chamber plate receiving part 200.

Further, the present invention provides a method of manufacturing amicro-chamber plate with a built-in sample, comprising a step S200 offorming a sample solution storing space between a cover 1310 formicro-chamber plate receiving part and an upper surface of amicro-chamber plate 100 for sample injection by closely contacting alower end of a cover 1310 for micro-chamber plate receiving part to theupper surface of the micro-chamber plate 100 for sample injection, thecover 1310 for micro-chamber plate receiving part comprising aprovisional storing part 1312 and an auxiliary covering part 1314connected with the provisional storing part 1312 and formed with athrough-hole 1314-1 for auxiliary covering part; and a step S300 ofmanufacturing a micro-chamber plate with a built-in sample by puttingthe micro-chamber plate 100 for sample injection and the cover 1310 formicro-chamber plate receiving part, which are closely contacted witheach other so as to form the sample solution space therebetween, into acentrifugal separator which can apply vacuum, applying centrifugal forceand injecting a sample solution provisionally stored in the provisionalstoring part 1312 into the micro-chamber plate 100 for sample injectionthrough a vessel communication part which is formed at the provisionalstoring part 1312 so as to be communicated with the sample solutionstoring space.

Preferably, the step S40, S300 of manufacturing the micro-chamber plate100A with the built-in sample comprises a vacuum and centrifugal forceapplying step of applying a vacuum into the centrifugal separator andgenerating first centrifugal force while the vacuum is applied into thecentrifugal separator; and a vacuum releasing and centrifugal forceapplying step of injecting the sample solution into the micro-chamberplate 100 for sample injection by releasing the vacuum in thecentrifugal separation while second centrifugal force larger than thefirst centrifugal force is generated by the centrifugal separator,wherein the first centrifugal force is a centrifugal force which cansuppress bumping of the sample solution, while the vacuum is appliedinto the centrifugal separator, and the vessel communication part is acutting line 312-1, 1312-1 which is formed at the provisional storingpart 312, 1312 so as to be opened by external force, and the secondcentrifugal force is a centrifugal force which can open the cutting line312-1, 1312-1.

Further, the present invention provides a method of manufacturing ananalytic micro-chamber plate using the micro-chamber plate with thebuilt-in sample manufactured by the above-mentioned method, comprising astep S50 of manufacturing the analytic micro-chamber plate by taking outthe micro-chamber plate 100A with the built-in sample from thecentrifugal separator and then sealing a separation membrane 130 of themicro-chamber plate 100A with the built-in sample.

Further, the present invention provides an analytic micro-chamber platein which a body sealing part 120 is formed at a lower surface thereof, asealed separation membrane is formed at an upper surface thereof, andthe unit number of chamber holes 112, which a sample solution includingnucleic acid and a special component 140 for analyzing the nucleic acidare built in, is formed, wherein the body sealing part 120 is formed ofa material that reflects light, and the sealed separation membrane is aseparation membrane 130 which is formed of a porous material and coatedand sealed with polymer oil so that a surface of the separation membrane130 has an increased optical transparency.

Further, the present invention provides an apparatus set formanufacturing a micro-chamber plate with a built-in sample, comprising amicro-chamber plate receiving part 200 which is formed with an upperopening; and a cover 310 for a micro-chamber plate receiving part, whichcomprises a provisional storing part 312 and an auxiliary covering part314 connected with the provisional storing part 312 and formed with athrough-hole 314-1 for auxiliary covering part, and which covers anupper opening of the micro-chamber plate receiving part 200, theprovisional storing part 312 being formed with a vessel communicationpart which can be opened and closed and is communicated with themicro-chamber plate receiving part 200 when being opened.

Preferably, the apparatus set further comprises a cover 320 forprovisional storing part which exposes the through-hole 314-1 forauxiliary covering part to an outside and partially closes theprovisional storing part 312, and a lower surface of the provisionalstoring part 312 is inserted into the micro-chamber plate receiving part200, and an upper end of the provisional storing part 312 and theauxiliary covering part 314 are disposed at an upper end of themicro-chamber plate receiving part 200, and also the apparatus setfurther comprises a cover 320 for provisional storing part which coversan upper end of the through-hole 314-1 for auxiliary covering part andan upper end of the provisional storing part 312, wherein the cover 320for provisional storing part is a membrane filter which allowspenetration of gas and prevents penetration of the sample solution, andthe vessel communication part is a cutting line 312-1 which is opened byexternal force.

Further, the present invention provides an apparatus set formanufacturing a micro-chamber plate with a built-in sample, comprising acover 1310 for micro-chamber plate receiving part, which comprises aprovisional storing part 1312 and an auxiliary covering part 1314connected with the provisional storing part 1312 and formed with athrough-hole 1314-1 for auxiliary covering part, and of which a lowerend is closely contacted with an upper surface of a micro-chamber plate100 for sample injection by coupling means so that a sample solutionstoring space S is formed between the cover 1310 for micro-chamber platereceiving part and the upper surface of the micro-chamber plate 100 forsample injection, wherein the provisional storing part 1312 is formedwith a vessel communication part which can be opened and closed and iscommunicated with the sample solution storing space when being opened.

Preferably, the coupling means comprises a micro-chamber plate receivingpart 1200 in which the micro-chamber plate 100 for sample injection issettled; and a coupling case 1400 of which an upper surface is formedwith the through-hole 1314-1 for auxiliary covering part and athrough-hole 1424 for case communicating the provisional storing part1312 to an outside, and which compresses an upper end of the cover 1310for micro-chamber plate receiving part so as to be coupled to themicro-chamber plate receiving part 1200, and the vessel communicationpart is a cutting line 1312-1 which is opened by external force, and acase cover 1500 is attached to the coupling case 1400 so as to cover thethrough-hole 1424 for case, such that the through-hole 1314-1 is exposedto an outside and the provisional storing part is closed partially, andthe case cover 1500 is attached to the coupling case 1400 so as to coverthe through-hole 1424, and the case cover 1500 is a membrane filterwhich allows penetration of gas and prevents penetration of the samplesolution.

According to the present invention as described above, since theseparation membrane which is the injection part of the sample solutionincluding nucleic acid is used as the optical measuring part, it ispossible to provide a simple structure, prevent measurement error of theoptical measuring part due to the contamination, reduce a size of theanalytic micro-chamber plate, facilely control the temperature and thusremarkably reduce the analyzing time.

Further, in case that the sample solution including nucleic acid isinjected into the chamber hole, since the gas in the chamber hole isfirstly removed by using vacuum, and then the injection of the samplesolution is performed through the separation membrane, it is possible tocompletely inject the sample solution within a short time without anyremained gas and prevent the error of the optically measured value dueto the remain gas. Further since the separation membrane is sealed withthe polymer oil such as mineral oil and silicon oil, it is possible toprevent the cross contamination due to the mixing of the solutions inthe chamber holes, thereby increasing the analyzing accuracy.

Further, since the multiple analytic micro-chamber plates can be formedintegrally, it is possible to compare and analyze various kinds ofsamples at the same time, thereby remarkably reducing the analyzingtime.

Further, since the separation membrane and the other surface of theoptical measuring part can be integrally formed with the analyticmicro-chamber plate, the analytic micro-chamber plate of the presentinvention can be manufactured by the compression molding of aluminum orthe like, and thus the production process and manufacturing cost can beremarkably reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentinvention will become apparent from the following description ofpreferred embodiments given in conjunction with the accompanyingdrawings, in which:

FIG. 1 is a flow chart of a first embodiment of the present invention.

FIG. 2 is a perspective view of a micro-chamber plate for sampleinjection, which is prepared by a manufacturing step of themicro-chamber plate for sample injection in FIG. 1.

FIG. 3 is a cross-sectional view taken along a line A-A′ of FIG. 2.

FIG. 4 is an exploded perspective view of a main part in FIG. 2.

FIG. 5 is a perspective view of an origin micro-chamber body prepared ina manufacturing step of the origin micro-chamber body in FIG. 1.

FIG. 6 is a cross-sectional view of the origin micro-chamber body inFIG. 5.

FIG. 7 is a cross-sectional view of explaining a coating step and acoupling step in FIG. 1.

FIG. 8 is a cross-sectional view of explaining a step of attaching abody sealing part in FIG. 1.

FIG. 9 is a cross-sectional view of explaining a special componentinjecting step in FIG. 1.

FIG. 10 is an exploded perspective view of explaining a step of settlingthe micro-chamber plate for sample injection, a step of disposing acovering part and a step of manufacturing a micro-chamber plate with abuilt-in sample.

FIG. 11 is an assembled perspective view of FIG. 10.

FIG. 12 is a cross-sectional view taken along a line B-B′ of FIG. 11, inwhich the micro-chamber plate for sample injection is settled.

FIG. 13 is an enlarged view of a cover for micro-chamber plate receivingpart in FIG. 10.

FIG. 14 is an enlarged view of a cover for provisional storing part inFIG. 10.

FIG. 15 is a perspective view of the covering part formed by couplingthe cover for the micro-chamber plate receiving part and the cover forprovisional storing part.

FIG. 16 is an enlarged view of the micro-chamber plate receiving part inFIG. 10.

FIG. 17 is a cross-sectional view of the micro-chamber plate with thebuilt-in sample, which is corresponding to FIG. 3.

FIG. 18 is a flow chart of a fifth embodiment of the present invention.

FIG. 19 is an exploded perspective view of explaining a step of forminga sample solution storing space and a step of manufacturing themicro-chamber plate with the built-in sample.

FIG. 20 is an assembled perspective view of FIG. 19.

FIG. 21 is a cross-sectional view taken along a line A-A′ of FIG. 20.

DETAILED DESCRIPTION OF MAIN ELEMENTS

100: micro-chamber plate for sample injection 100A: micro-chamber platewith a built-in sample 112: chamber hole 120: body sealing part 130: aseparation membrane 140: special component 200: micro-chamber platereceiving part 300: covering part 310: a cover for micro-chamber platereceiving part 312: provisional storing part 312-1: cutting line 314:auxiliary covering part 314-1: through-hole for auxiliary covering part320: a cover for provisional storing part 1200: micro-chamber platereceiving part 1310: a cover for micro-chamber plate receiving part1312: provisional storing part 1312-1: cutting line 1314: auxiliarycovering part 1314-1: through-hole for auxiliary covering part 1400:coupling case 1424: through-hole of the case 1500: case cover S: samplesolution storing space

DETAILED DESCRIPTION

Hereinafter, the embodiments of the present invention will be describedin detail with reference to accompanying drawings.

First Embodiment

The first embodiment relates to a method of manufacturing an analyticmicro-chamber plate according to the present invention.

Referring to FIG. 1, the first embodiment includes a step S10 ofmanufacturing a micro-chamber plate for sample injection, a step S20 ofsettling the micro-chamber plate for sample injection, a step S30 ofdisposing a covering part, a step S40 of manufacturing a micro-chamberplate with a built-in sample, and a step S50 of manufacturing ananalytic micro-chamber plate.

1. Step S10 of Manufacturing the Micro-Chamber Plate for SampleInjection

Referring to FIGS. 2 to 4, in the step S10 of manufacturing themicro-chamber plate for sample injection, the micro-chamber plate 100for sample injection, in which a special component 140 is built in, ismanufactured. The micro-chamber plate 100 for sample injection includesa micro-chamber body 110, a body sealing part 120 and a separationmembrane 130.

Therefore, referring to FIGS. 1 to 4, the step S10 of manufacturing themicro-chamber plate for sample injection includes a micro-chamber bodypreparing step S11 which prepares the micro-chamber body 110, a bodysealing part attaching step S12 which forms the body sealing part 120 ata lower surface of the micro-chamber body 110, a special componentinjecting step S13 which injects the special component 140, and aseparation membrane attaching step S14 which forms the separationmembrane 130 at an upper surface of the micro-chamber body 110.

Meanwhile, referring to FIG. 1, the micro-chamber body preparing stepS11 includes a origin micro-chamber body preparing step S11-1, a coatingstep S11-2 and a coupling step S11-3.

Referring to FIGS. 5 and 6, in the origin micro-chamber body preparingstep S11-1, an origin micro-chamber body 110-1 which is formed with aunit number of origin chamber holes 110-1H passing through upper andlower surfaces thereof is prepared. The origin micro-chamber body 110-1is formed of a material, preferably, which has durability against heatapplied in a polymerase chain reaction (PCR) or other analyzingreaction, more preferably, which is not deformed at 0˜100° C. Therefore,the origin micro-chamber body 110-1 may be formed of an aluminum,silicon wafer, glass, metallic or plastic material. Meanwhile, aplurality of the origin micro-chamber bodies 110-1 may be connected witheach other through connecting pieces (not designated by a referencenumeral). A reference numeral 110-1S is an origin micro-chamber body setformed by connecting the multiple origin micro-chamber bodies 110-1.

Referring to FIG. 7, the micro-chamber body 110 is manufactured throughthe coating step S11-2 and the coupling step S11-3. In the micro-chamberbody 110, the unit number of chamber holes 112 corresponding to the unitnumber of origin chamber holes 110-1H are formed. Each chamber hole110-1H may be formed to have a width of 0.3 to 3 mm and a depth of 0.5to 5 mm. In case of the first embodiment, since multiple chamber holes112 are formed in one micro-chamber body 110, it is possible toquantitatively analyze a large number of nucleic acids at the same time,and since the chamber hole 112 has a shallow depth, it is possible toprovide high thermal conduction performance, reduce an analyzing timeand also improve analyzing accuracy.

Referring to FIG. 7, in the coating step S11-2, the origin micro-chamberbody 110-1 is dipped in a polymer solution, and thus a polymer coatinglayer 110-2 is formed on a surface of the origin micro-chamber body110-1 and inner surfaces of the unit number of origin chamber holes110-1H (referring to FIG. 6).

In the embodiment, an aqueous solution in which ES-120s (which wascopolymer polyester obtained by reacting complex aromatic dicarboxylicacid and complex aliphatic diol) resin, as a polyester-based resin,manufacture by Basekorea co., Ltd. was diluted to 50% with toluene (4)and MEK (1) was diluted to 5˜20 vol % using toluene so as to have anadjusted viscosity. Then, the origin micro-chamber body 110-1 was coatedwhile changing the dipping number thereof from one time to three times.In order to prevent clogging of the origin chamber hole 110-1H and alsoobtain the uniform polymer coating layer 110-2, it was the mostpreferable that the viscosity was 5 to 10 vol % and the dipping numberwas two times. Meanwhile, in case that the origin micro-chamber body110-1 is formed of aluminum, it is preferable that a white anodizingstep in which the surface of the origin micro-chamber body 110-1 iswhite-anodized is carried out before the coating step S11-2.

Referring to FIG. 7, in the coupling step S11-3, a surface of thepolymer coating layer 110-2 is treated by a coupling process. Thecoupling process is to remove carboxylic acid as a functional groupwhich is present in the polyester-based resin.

In the embodiment, the coupling process was carried out under an ethanolsolvent using 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC) and 4-Dimethylaminopyridine (DMAP). First of all,DMAP and EDC having a concentration of 0.25M were prepared respectively,and 50 ml of DMAP and 9 ml of EDC were mixed and reacted for 12 hours ormore at room temperature. And then three times of cleaning processes areperformed using ethanol and distilled water, respectively, and thecoupling process was completed by desiccation.

Since the polyester-based resin used in the present invention iscolorless and transparent and also has very excellent adhesive propertywith respect to an aluminum surface, it is estimated that thepolyester-based resin is the most suitable.

Referring to FIG. 8, in the body sealing part attaching step S12, loweropenings of the unit number of chamber holes 112 are sealed by the bodysealing part 120 after the coupling step S11-3. In the body sealing partattaching step S12, the body sealing part 120 is contacted with thepolymer coating layer 110-2 and then pressed at a high temperature so asto be attached to the polymer coating layer 110-2. The body sealing part120 may be formed of a material, which can reflect light to theseparation membrane 130 (referring to FIG. 17), in order to facilelyirradiate light through the separation membrane 130 (referring to FIG.17) upon the optical measurement and then facilely measure excitationlight generated from the sample. Therefore, in case that the loweropenings of the unit number of chamber holes 112 are sealed with atransparent film, the body sealing part 120 may further include areflecting film attached to an outer surface of the transparent film.Meanwhile, in case that the body sealing part 120 is formed of othermaterial which is not transparent or does not reflect light, areflecting layer may be further provided on a surface of the bodysealing part 120, which is corresponding to the lower openings of theunit number of chamber holes 112. Meanwhile, the body sealing part 120is formed of a material which can prevent leakage of a sample solutionincluding nucleic acid.

In the embodiment, a product of a transparent or opaque white polymerfilm type was used as the body sealing part 120. For example, atransparent film formed of a PET (polyethylene terephthalate) materialand having a thickness of 40 μm and an opaque white film (brand name:ST-DF-50W), which were manufacture by KM industrial co., Ltd., were usedall. By using these, it was possible to obtain more precise opticalmeasurement values.

Referring to FIG. 9, in the special component injecting step S13, thespecial component 140 for analyzing nucleic acid containing a primer ora probe is injected into each of the unit number of chamber holes 112 ofwhich the lower openings are sealed by the body sealing part 120. Thespecial component 140 may further include a fluorescence analyzing agentfor analyzing nucleic acid, and if necessary, a nucleic acid amplifyingenzyme, dNTP, a buffer or a stabilizer (e.g., polyol, carbohydrate, soalbumin or the like which is molecularly mixed well with the reactionsolution, primer, enzyme or the like, thereby stabilizing them andreducing attachment to the vessel). The special component 140 may beused in a dried, semi-dried or liquefied state according to itscomposition.

Referring to FIG. 3, in the separation membrane attaching step S14, theseparation membrane 130 is attached to an upper surface of themicro-chamber body 110. That is, in the separation membrane attachingstep S14, the separation membrane 130 is attached to upper end sides ofthe unit number of chamber holes 112 so as to cover upper openings ofthe unit number of chamber holes 112 in which the special component 140is injected. The separation membrane 130 is formed so as to preventpenetration of the special component 140 but allow penetration of thesample solution. Therefore, the special component 140 is prevented frombeing leaked from an inner side of the chamber hole 112 to an outsidethrough the separation membrane 130, but the sample solution includingnucleic acid can be introduced from an outside into the chamber hole 112through the separation membrane 130. Therefore, the separation membrane130 may be formed of a porous material which prevents the penetration ofthe special component 140 but allows the penetration of the samplesolution including nucleic acid. In case that the separation membrane130 is formed of a porous material, the separation membrane 130 may besealingly coated with polymer oil so as to improve an opticaltransparency of the separation membrane 130, thereby facilitating theoptical measurement with respect to the sample in the chamber hole 112.The polymer oil may be mineral oil, silicone oil, hydrocarbon oil,paraffin wax or the like. The separation membrane 130 formed of theporous material may be formed of a micro-pore shape, a mesh shape or anunwoven shape. Preferably, the porous material has a pore size of 0.1 to100 μm. The porous separation membrane 130 may be a polymer membrane.Meanwhile, the separation membrane 130 is pressed at a high temperaturewhile being contacted with the polymer coating layer 110-2 and thusattached to the polymer coating layer 110-2.

In the embodiment, a great number of porous materials having desiredpore sizes were used as the separation membrane 130. More detailedly, aproduct formed of a PC (polycarbonate) material and having a pore sizeof 12 μm, which was manufacture Whatman company was used. This wascaused by that the sample injection was facile due to its large poresize and the transparency was increased by the mineral oil, therebyfacilitating the optical measurement.

Meanwhile, in other embodiment, the separation membrane 130 may beformed of a film which can be punched. This type of separation membranemay be formed of Teflon, polypropylene, polyethylene, polyester orpolyvinyl chloride. 1 to 10 hollowed portions may be formed per onechamber hole 112. In order to prevent leakage of the built-in specialcomponent 140 and facilitate injection of the sample solution includingnucleic acid, the punched portion may have a width of 10 μm to 1 mm or100 to 500 μm. Meanwhile, since the film type separation membrane has agood optical transparency, it may make the optical measurement possible.

2. Step S20 of Settling the Micro-Chamber Plate for Sample Injection

Referring to FIGS. 10 to 12, in the step S20 of settling themicro-chamber plate for sample injection, the micro-chamber plate 100for sample injection is settled at a micro-chamber plate receiving part200 formed with an upper opening. A reference numeral 200S is amicro-chamber plate receiving part set in which multiple micro-chamberplate receiving parts 200 are connected with each other.

3. Step S30 of Disposing a Covering Part

Referring to FIG. 1, the step S30 of disposing the covering partincludes a step S31 of preparing a cover for micro-chamber platereceiving part, a step S32 of preparing a cover for provisional storingpart, a step S33 of attaching the cover for provisional storing part anda step S34 of provisionally storing a sample solution.

Referring to FIG. 13, in the step S31 of preparing a cover formicro-chamber plate receiving part, 312, a cover 310 for micro-chamberplate receiving part, in which a provisional storing part 312 and anauxiliary covering part 314 are formed integrally, is prepared. Areference numeral 310S is a cover set for micro-chamber plate receivingpart, in which multiple covers 310 for micro-chamber plate receivingpart are connected with each other.

Referring to FIG. 13, the provisional storing part 312 is a vessel whichprovisionally stores the sample solution including nucleic acid, and avessel communication part is formed at a lower surface thereof. Thevessel communication part may be a cutting line 312-1 which is opened byexternal force. Therefore, if the external force is not applied to thelower surface of the provisional storing portion 312, it is preventedthat the sample solution including nucleic acid which is provisionallystored in the provisional storing part 312 is leaked to an outside ofthe provisional storing part 312. The provisional storing part 312 maybe formed of a silicon material. Meanwhile, the cutting line 312 may bein the formed of “+” shape, “<” shape, “=” shape or “x” shape.

Referring to FIG. 13, the auxiliary covering part 314 is formed into aplate shape which is connected to an upper end of a circumferentialsurface of the provisional storing part 312 so as to be arrangedhorizontally. The auxiliary covering part 314 is formed with athrough-hole 314-1 for auxiliary covering part, which passes throughupper and lower surfaces of the auxiliary covering part 314.

Referring to FIG. 14, in the step S32 of preparing a cover forprovisional storing part, a cover 320 for provisional storing partformed with a through-hole 324 for provisional storing part cover isprepared. The cover 320 for provisional storing part may be formed intoa thin film shape. A reference numeral 320S is a cover set forprovisional storing part, in which multiple covers 320 for provisionalstoring part are connected with each other.

Referring to FIG. 15, the cover 320 for provisional storing part isformed so that the through-hole 314-1 for auxiliary covering part isexposed to an outside and an upper portion of the provisional storingpart 312 is partially closed when the cover 320 for provisional storingpart is attached to an upper end of the cover 310 for micro-chamberplate receiving part. In this case, the rest of the upper portion of theprovisional storing part 312 is exposed to the outside through thethrough-hole 324 for provisional storing part cover.

Referring to FIG. 15, in the step S33 of attaching the cover forprovisional storing part, the cover 320 for provisional storing part isattached to the upper end of the cover 310 for micro-chamber platereceiving part. In order to attach the cover 320 for provisional storingpart, an adhesive may be previously applied to the cover 310 formicro-chamber plate receiving part. The adhesive may be a polymeradhesive, a double-sided tape or the like. Therefore, the upper portionof the provisional storing part 312 is closed partially, and thethrough-hole 314-1 for auxiliary covering part and the rest of the upperportion of the provisional storing part 312 are exposed to the outsidethrough the through-hole 324 for provisional storing part cover. Acovering part 300 is formed by performing the step S33 of attaching thecover for provisional storing part.

Referring to FIGS. 11, 15 and 16, if the step S33 of attaching the coverfor provisional storing part is carried out, the covering part 300 issettled on an upper end of the micro-chamber plate receiving part 200while covering an upper opening of the micro-chamber plate receivingpart 200.

Referring to FIG. 12, if the covering part 300 is settled on an upperend of the micro-chamber plate receiving part 200, a lower surface ofthe provisional storing part 312 is inserted into the micro-chamberplate receiving part 200, and an upper end of the provisional storingpart 312 and the auxiliary covering part 314 are disposed on an upperend of the micro-chamber plate receiving part 200.

Referring to FIG. 12, if the covering part 300 is settled on the upperend of the micro-chamber plate receiving part 200, an inner side of themicro-chamber plate receiving part 200 is communicated with the outsidethrough the through-hole 314-1 for auxiliary covering part and thethrough-hole 324 for provisional storing part cover, and the provisionalstoring part 312 is communicated with the outside through thethrough-hole 324 for provisional storing part cover.

Referring to FIG. 12, in the step S34 of provisionally storing thesample solution, the sample solution including nucleic acid isprovisionally stored in the provisional storing part 312 through thethrough-hole 324 of provisional storing part cover.

4. Step S40 of Manufacturing a Micro-Chamber Plate with a Built-inSample

Referring to FIG. 1, the step S40 of manufacturing the micro-chamberplate with the built-in sample includes a step S41 of applying vacuumand centrifugal force and a step S42 of releasing vacuum and applyingcentrifugal force.

In the step S41 of applying the vacuum and centrifugal force, first ofall, the micro-chamber plate receiving part 200 on which the coveringpart 300 is disposed is put into a centrifugal separator which can applyvacuum. In this case, referring to FIG. 11, the through-hole 324 forprovisional storing part cover is directed upward, and the cover 320 forprovisional storing part is directed to a rotational center of thecentrifugal separator, and the lower surface of the micro-chamber platereceiving part 200 is directed to an opposite side of the rotationalcenter of the centrifugal separator. Then, the vacuum is applied to thecentrifugal separator, and the centrifugal separator is operated so thatfirst centrifugal force is applied to the micro-chamber plate receivingpart 200. If the centrifugal force is not applied in the vacuum state, aboiling point of the sample solution including nucleic acid is lowered,and bumping phenomenon is occurred, thereby resulting in contamination.Therefore, the first centrifugal force functions to suppress the bumpingphenomenon while the vacuum is applied in the centrifugal separator.Further, the cutting line 312-1 is not opened by the first centrifugalforce, and thus it is prevented that the cutting line 312-1 is opened bythe first centrifugal force and the solution is contacted with theseparation membrane 130.

In the step S42 of releasing the vacuum and applying the centrifugalforce, second centrifugal force generated from the centrifugal separatoris larger than the first centrifugal force, such that the cutting line312-1 is opened by the second centrifugal force. Then, the vacuumapplied in the centrifugal separator is released, and the samplesolution is injected into the micro-chamber plate 100 for sampleinjection through the cutting line 312-1 and the separation membrane130. Therefore, as shown in FIG. 17, the sample solution is injected inthe chamber hole 112, and thus a micro-chamber plate 100A with abuilt-in sample which is mixed with the special component 140 (referringto FIG. 3) is prepared. A reference numeral 150 is a mixed solution ofthe special component 140 (referring to FIG. 3) and the sample solution.

According to the embodiment, in the step S41 of applying the vacuum andcentrifugal force, the first centrifugal force is maintained to be lessthan 42 g, and the bumping of the sample solution is suppressed.

In the step S42 of releasing the vacuum and applying the centrifugalforce, the centrifugal force is gradually increased and maintained to bemore than 242 g, and the sample solution is injected for 1 minute intothe micro-chamber plate 100 for sample injection while the vacuum isreleased. Therefore, the sample solution can be completely injected intothe chamber hole 112.

A reason why the sample solution is injected while the vacuum isreleased is as follows: if the sample solution is contacted with theseparation membrane 130 while the vacuum is applied, it is impossible tocompletely inject the sample solution due to the property of theseparation membrane 130.

5. Step S50 of Manufacturing a Micro-Chamber Plate

In the step S50 of manufacturing a micro-chamber plate, first of all,the micro-chamber plate 100A with the built-in sample (referring to FIG.17) is taken out of the centrifugal separator. Then, a surface of theseparation membrane 130 of the micro-chamber plate 100A with thebuilt-in sample (referring to FIG. 17) is sealed so that the samplesolution built in the micro-chamber plate 100A with the built-in sample(referring to FIG. 17) is not leaked to the outside upon an analyzingreaction including the PCR reaction. Further, the sealing of the surfaceof the separation membrane 130 is performed so that the opticaltransparency of the separation membrane 130 is increased and thus theoptical measurement with respect to the sample in the chamber hole 112can be facilely performed through the separation membrane 130.

By performing the step S50 of manufacturing a micro-chamber plate, ananalytic micro chamber plate which can be used in the analyzing reactionincluding the PCR is manufactured.

Since the special component 140 including the primer or probe is builtin the analytic micro chamber plate, the analytic micro chamber platecan be used in a real-time PCR, and it can be also used in a fixedtemperature enzyme reaction and an LCR (Ligase Chain Reaction). Further,it can be used variously by changing the special component 40 or thelike.

In case of the first embodiment, since the separation membrane 130 isformed of the porous material, the surface of the separation membrane130 of the micro-chamber plate 100A with the built-in sample is coatedand sealed with the polymer oil. The polymer oil may be mineral oil,silicone oil, hydrocarbon oil, paraffin wax or the like. In case thatthe separation membrane 130 is a polypropylene membrane, it may becoated and sealed with the mineral oil.

If the separation membrane 130 which is the polypropylene membrane iscoated and sealed with the mineral oil, the hydrophobic mineral oilpushes the sample including water penetrated through the hydrophobicpolypropylene membrane and then occupies the place due tohydrophilicity-hydrophobicity effect. Meanwhile, since the mineral oilhas a density similar to the polypropylene membrane, the opticaltransparency of the separation membrane 130 is increased and the opticalmeasurement with respect to the sample in the chamber hole 112 isfacilitated, and since the polypropylene membrane is sealed with themineral oil, it is prevented that the sample in the chamber hole 112 isleaked and evaporated.

Meanwhile, in other embodiment, if the separation membrane 130 is formedof the film which can be punched, the surface of the separation membrane130 of the micro-chamber plate 100A with the built-in sample (referringto FIG. 17) may be sealed with an adhesive film (tape).

Meanwhile, in case of other embodiment, the cover 320 for provisionalstoring part may be a membrane filter which allows the penetration ofgas but prevents the penetration of the sample solution. In order toattach the membrane filter as the cover 320 for provisional storingpart, an adhesive may be previously applied to the cover 310 formicro-chamber plate receiving part. The adhesive may be a polymeradhesive, a double-sided tape or the like. In case that the cover 320for provisional storing part is attached to the upper end of the cover310 for micro-chamber plate receiving part, the cover 320 forprovisional storing part is formed to cover the upper end of thethrough-hole 314-1 for auxiliary covering part and the upper end of theprovisional storing part 312. In this case, the step S34 ofprovisionally storing the sample solution is carried out before the stepS33 of attaching the cover for provisional storing part.

Meanwhile, in case of other embodiment, it may not include the step S32of preparing a cover for provisional storing part and the step S33 ofattaching the cover for provisional storing part. In this case, thecovering part 300 may be the cover 310 for micro-chamber plate receivingpart. Therefore, before the step S41 of applying the vacuum andcentrifugal force, a bottom surface of the provisional storing part 312is directed downward (not gravity direction) so that the sample solutionis not leaked through the opened upper portion of the provisionalstoring part 312. As the step S41 of applying the vacuum and centrifugalforce is carried out, the opened upper portion of the provisionalstoring part 312 is directed to the rotational center of the centrifugalseparator, and the lower surface of the provisional storing part 312,i.e., the cutting line 312-1 is directed to an opposite side of therotational center of the centrifugal separator.

Meanwhile, in case of other embodiment, the cover 320 for provisionalstoring part may be formed to cover the through-hole 314-1 for auxiliarycovering part and the provisional storing part 312. In this case, thecover 320 for provisional storing part is the membrane filter whichallows the penetration of gas but prevents the penetration of the samplesolution. In this case, the cover 320 for provisional storing part isnot formed with the through-hole 324 for provisional storing part cover.

Second Embodiment

The second embodiment relates to a method of manufacturing themicro-chamber plate with a built-in sample according to the presentinvention.

The second embodiment includes a step S10 of manufacturing amicro-chamber plate for sample injection, a step S20 of settling amicro-chamber plate for sample injection, a step S30 of disposing acovering part, and a step S40 of manufacturing a micro-chamber platewith the built-in sample which are described in the first embodiment.The description thereof is based on that of the first embodiment.

Third Embodiment

The third embodiment relates to an analytic micro-chamber plate.

The analytic micro-chamber plate (not shown) is the same as themicro-chamber plate 100A with the built-in sample (referring to FIG. 17)except the separation membrane 130, and thus it will be described withreference with FIG. 17.

Referring to FIGS. 6 and 17, the third embodiment includes an originmicro-chamber body 110-1 in which the unit number of chamber holes110-1H are formed to pass through upper and lower surfaces thereof.

Referring to FIGS. 7 and 17, a polymer coating layer 110-2 is formed ona surface of the origin micro-chamber body 110-1 and inner surfaces ofthe unit number of origin chamber holes 110-1H (referring to FIG. 6).The unit number of chamber holes 112 corresponding to the unit number oforigin chamber holes 110-1H are formed by the polymer coating layer110-2.

Referring to FIGS. 8 and 17, a body sealing part 120 is formed at alower surface of the micro-chamber body 110 so as to seal lower openingsof the unit number of chamber holes 112. The body sealing part 120 is toprevent leakage of the sample solution including nucleic acid.Meanwhile, the body sealing part 120 is formed of a PET (polyethyleneterephthalate) material which reflects light.

Referring to FIG. 17, a sealed separation membrane (not shown) forcovering the upper openings of the unit number of chamber holes 112 isformed at an upper surface of the micro-chamber body 110. The sealedseparation membrane (not shown) is formed by coating and sealing aseparation membrane 130 formed of a porous material with polymer oil.The polymer oil may be mineral oil, silicone oil, hydrocarbon oil,paraffin wax or the like. Meanwhile, by coating the separation membrane130 formed of the porous material with the mineral oil, the opticaltransparency is increased. The separation membrane 130 may be apolypropylene membrane.

Referring to FIG. 17, the sample solution including nucleic acid and aspecial component 140 for analyzing the nucleic acid including a primeror a probe are built in each of the unit number of chamber holes 112. Areference numeral 150 is a mixed solution thereof.

Other elements which are not described are based on the description inthe first embodiment.

Fourth Embodiment

The fourth embodiment relates to an apparatus set for manufacturing amicro-chamber plate with a built-in sample according to the presentinvention.

Referring to FIGS. 10 and 12, the fourth embodiment includes amicro-chamber plate receiving part 200 in which the micro-chamber plate100 for sample injection can be settled. An upper opening which settlesthe micro-chamber plate 100 for sample injection is formed at an upperend of the micro-chamber plate receiving part 200.

Referring to FIGS. 10 and 12, a cover 310 for micro-chamber platereceiving part which covers the upper openings of the micro-chamberplate receiving part 200 is settled at an upper portion of themicro-chamber plate receiving part 200.

Referring to FIGS. 12 and 15, the cover 310 for micro-chamber platereceiving part includes a provisional storing part 312 in which thesample solution can be provisionally stored, and a plate-shapedauxiliary covering part 314 which is formed to be connected with acircumferential surface of the provisional storing part 312.

Referring to FIG. 13, a vessel communication part is formed at a lowersurface of the provisional storing part 312. The vessel communicationpart may be a cutting line 312-1 which is opened by external force.Meanwhile, the auxiliary covering part 314 is formed with a through-hole314-1 for auxiliary covering part, which passes through upper and lowersurfaces of the auxiliary covering part 314.

Referring to FIGS. 11 and 12, a lower surface of the provisional storingpart 312 is inserted into the micro-chamber plate receiving part 200,and an upper end of the provisional storing part 312 and the auxiliarycovering part 314 are disposed on an upper end of the micro-chamberplate receiving part 200.

Referring to FIGS. 12 and 15, a cover 320 for provisional storing partis attached to an upper end of the cover 310 for micro-chamber platereceiving part. The cover 320 for provisional storing part is attachedso that the upper portion of the provisional storing part 312 is closedpartially, and the through-hole 314-1 for auxiliary covering part andthe rest of the upper portion of the provisional storing part 312 areexposed to the outside through the through-hole 324 for provisionalstoring part cover.

Other elements which are not described are based on the description inthe first embodiment.

Fifth Embodiment

The fifth embodiment relates to a method of manufacturing an analyticmicro-chamber plate according to the present invention.

Referring to FIG. 18, the fifth embodiment includes a step S100 ofpreparing a micro-chamber plate for sample injection, a step S200 offorming a sample solution storing space, a step S300 of preparing amicro-chamber plate with a built-in sample, and a step S400 of preparingan analytic micro-chamber plate.

Among them, the step S100 of preparing the micro-chamber plate forsample injection and the step S400 of preparing the analyticmicro-chamber plate are based on the description in the firstembodiment.

1. Step S200 of Forming a Sample Solution Storing Space

Referring to FIG. 18, the step S200 of forming the sample solutionstoring space includes a step S210 for preparing a micro-chamber platereceiving part, a step S220 for preparing a cover for micro-chamberplate receiving part, a step S230 of preparing a coupling case, a stepS240 of coupling the case, and a step S250 of attaching a case cover.

Referring to FIGS. 19 and 21, in the step S210 for preparing themicro-chamber plate receiving part, a micro-chamber plate receiving part1200 is formed. The micro-chamber plate receiving part 1200 may beformed into a flat plate shape. The micro-chamber plate receiving part1200 is to settle the micro-chamber plate for sample injection. Areference numeral 1200S is a micro-chamber plate receiving part set inwhich multiple micro-chamber plate receiving parts 1200 are formedintegrally. Meanwhile, a coupling protrusion 1200S-1 is formed at a sidesurface of the micro-chamber plate receiving part set 1200S.

Referring to FIGS. 19 and 21, in the step S220 for preparing the coverfor micro-chamber plate receiving part, a cover 1310 for micro-chamberplate receiving part, in which a provisional storing part 1312 and anauxiliary covering part 1314 are formed integrally, is prepared. Areference numeral 1310S in FIG. 19 is a cover set for micro-chamberplate receiving part, in which multiple covers 1310 for micro-chamberplate receiving part are connected with each other.

Referring to FIGS. 19 and 21, the provisional storing part 1312 is avessel which provisionally stores the sample solution including nucleicacid, and a vessel communication part is formed at a lower surfacethereof. The vessel communication part may be a cutting line 1312-1which is opened by external force. The description of the cutting line1312-1 is based on that in the first embodiment.

Referring to FIGS. 19 and 21, the auxiliary covering part 1314 is formedinto a protrusion shape or a plate shape, like in the first embodiment,which is connected to a circumferential surface of the provisionalstoring part 1312 so as to be arranged horizontally. The auxiliarycovering part 1314 is formed with a through-hole 1314-1 for auxiliarycovering part, which passes through upper and lower surfaces of theauxiliary covering part 1314.

Referring to FIGS. 19 and 21, a ring-shaped cover supporting part 1316is formed to be protruded from a lower edge of the cover 1310 formicro-chamber plate receiving part. The cover supporting part 1316 isformed so that a lower end of the through-hole 1314-1 for auxiliarycovering part is located in the cover supporting part 1316.

Referring to FIGS. 19 and 21, in the step S230 of preparing a couplingcase, a coupling case 1400 which is formed with a through-hole 1424 forcase is formed. The through-hole 1424 for case is formed to becommunicated with the through-hole 1314-1 for auxiliary covering partand the provisional storing part 1312 when the coupling case 1400 issettled at the cover 1310 for micro-chamber plate receiving part. Areference numeral 1400S in FIG. 19 is a coupling case set in whichmultiple coupling cases 1400 are connected with each other. Meanwhile, acase coupling groove 1400S-1 in which the coupling protrusion 1200S-1 isinserted is formed at a side surface of the coupling case set 1400S.

Referring to FIGS. 19 and 21, in the step S240 of coupling the case, thecoupling case 1400 is pressed to an upper end of the cover 1310 formicro-chamber plate receiving part and then coupled with themicro-chamber plate receiving part 1200. The coupling between thecoupling case 1400 and the micro-chamber plate receiving part 1200 isachieved by inserting the coupling protrusion 1200S-1 into the casecoupling groove 1400S-1. Since the coupling case 1400 is coupled to themicro-chamber plate receiving part 1200, a lower end of the coversupporting part 1316 is closely contacted with an upper surface of themicro-chamber plate 100 for sample injection, and thus a sample solutionstoring space S is formed between the cover 1310 for micro-chamber platereceiving part and the micro-chamber plate 100 for sample injection. Asthe step S240 of coupling the case is performed, a lower end of thethrough-hole 1314-1 for auxiliary covering part is communicated with thesample solution storing space S, and the through-hole 1424 for case iscommunicated with the through-hole 1314-1 for auxiliary covering partand the provisional storing part 1312, respectively.

Referring to FIGS. 19 and 21, in the step S250 of attaching the casecover, a case cover 1500 is attached to the coupling case 1400. In orderto attach the case cover 1500, an adhesive may be previously applied tothe coupling case 1400. The adhesive may be a polymer adhesive, adouble-sided tape or the like. The case cover 1500 is formed with athrough-hole 1524 for case cover which passes through upper and lowersurfaces thereof. The step S250 of attaching the case cover is performedso that the through-hole 1524 for case cover exposes the through-hole1314-1 for auxiliary covering part to an outside and partially closesthe provisional storing part 1312. A reference numeral 1500S in FIG. 19is a case cover set in which multiple case covers 1500 are connectedwith each other.

Meanwhile, after performing the step S250 of attaching the case cover,the sample solution including nucleic acid is provisionally stored inthe provisional storing part 1312 through the through-hole 1524 for casecover.

2. Step S300 of Preparing a Micro-Chamber Plate with a Built-in Sample

The step S300 of preparing the micro-chamber plate with the built-insample includes a step of applying vacuum and centrifugal force and astep of releasing vacuum and applying centrifugal force, like in thefirst embodiment.

In the step of applying the vacuum and centrifugal force, themicro-chamber plate 100 for sample injection and the cover 1310 formicro-chamber plate receiving part are put into a centrifugal separatorwhich can apply vacuum. In this case, referring to FIG. 21,

the through-hole 1524 for case cover is directed upward, and the casecover 1500 is directed to a rotational center of the centrifugalseparator, and a bottom surface of the provisional storing part 1312 isdirected to an opposite side of the rotational center of the centrifugalseparator. Other elements are based on the description of the firstembodiment.

Meanwhile, in case of other embodiment, the case cover 1500 which coversthe through-hole 1424 for case is attached to the coupling case 1400.The case cover 1500 is formed into a membrane filter which allowspenetration of gas but prevents penetration of the sample solution. Inthis case, the case cover 1500 is not formed with the through-hole 1424for case. Further, in order to attach the membrane filter as the casecover 1500, an adhesive may be applied to the coupling case 1400. Theadhesive may be a polymer adhesive, a double-sided tape or the like.

Meanwhile, in case of other embodiment, the case cover 1500 may be notattached to the coupling case 1400.

Other elements are based on the description of the first embodiment.

Sixth Embodiment

The sixth embodiment relates to a method of manufacturing amicro-chamber plate with a built-in sample.

The sixth embodiment includes a step S100 of preparing a micro-chamberplate for sample injection, a step S200 of forming a sample solutionstoring space, and a step S300 of preparing a micro-chamber plate with abuilt-in sample, which are described in the fifth embodiment.

Seventh Embodiment

The seventh embodiment relates to an apparatus set for manufacturing amicro-chamber plate with a built-in sample.

Referring to FIGS. 19 and 21, the seventh embodiment includes amicro-chamber plate receiving part 1200. The micro-chamber platereceiving part 1200 may be formed into a flat plate shape. Themicro-chamber plate receiving part 1200 is to settle the micro-chamberplate 100 for sample injection. A reference numeral 1200S is amicro-chamber plate receiving part set in which multiple micro-chamberplate receiving parts 1200 are connected with each other. Meanwhile, acoupling protrusion 1200S-1 is formed at a side surface of themicro-chamber plate receiving part set 1200S.

Referring to FIGS. 19 and 21, the seventh embodiment includes a cover1310 for micro-chamber plate receiving part in which a provisionalstoring part 1312 and an auxiliary covering part 1314 are formedintegrally. A reference numeral 1310S in FIG. 19 is a cover set for amicro-chamber plate receiving part in which multiple covers for amicro-chamber plate receiving part are connected with each other.

Referring to FIGS. 19 and 21, the provisional storing part 1312 is avessel which provisionally stores the sample solution including nucleicacid, and a vessel communication part is formed at a lower surfacethereof. The vessel communication part may be a cutting line 1312-1which is opened by external force. The description of the cutting line1312-1 is based on that in the first embodiment.

Referring to FIGS. 19 and 21, the auxiliary covering part 1314 is formedinto a protrusion shape or a plate shape, like in the first embodiment,which is connected to a circumferential surface of the provisionalstoring part 1312 so as to be arranged horizontally. The auxiliarycovering part 1314 is formed with a through-hole 1314-1 for auxiliarycovering part, which passes through upper and lower surfaces of theauxiliary covering part 1314.

Referring to FIGS. 19 and 21, a ring-shaped cover supporting part 1316is formed to be protruded from a lower edge of the cover 1310 formicro-chamber plate receiving part. The cover supporting part 1316 isformed so that a lower end of the through-hole 1314-1 for auxiliarycovering part is located in the cover supporting part 1316.

Referring to FIGS. 19 and 21, the seventh embodiment includes a couplingcase 1400. The coupling case 1400 is formed with an through-hole 1424for case, which passes through upper and lower surfaces of the couplingcase 1400. The through-hole 1424 for case is formed to be communicatedwith the through-hole 1314-1 for auxiliary covering part and theprovisional storing part 1312 when the coupling case 1400 is settled atthe upper end of the cover 1310 for micro-chamber plate receiving part.A reference numeral 1400S in FIG. 19 is a coupling case set in whichmultiple coupling cases 1400 are connected with each other. Meanwhile, acase coupling groove 1400S-1 in which the coupling protrusion 1200S-1 isinserted is formed at a side surface of the coupling case set 1400S.

Referring to FIGS. 19 and 21, the coupling case 1400 is pressed to anupper end of the cover 1310 for micro-chamber plate receiving part andthen coupled with the micro-chamber plate receiving part 1200. Thecoupling between the coupling case 1400 and the micro-chamber platereceiving part 1200 is achieved by inserting the coupling protrusion1200S-1 into the case coupling groove 1400S-1. Since the coupling case1400 is coupled to the micro-chamber plate receiving part 1200, a lowerend of the cover supporting part 1316 is closely contacted with an uppersurface of the micro-chamber plate 100 for sample injection, and thus asample solution storing space S is formed between the cover 1310 formicro-chamber plate receiving part and the micro-chamber plate 100 forsample injection. As the coupling case 1400 is coupled with themicro-chamber plate receiving part 1200, a lower end of the through-hole1314-1 for auxiliary covering part is communicated with the samplesolution storing space S, and the through-hole 1424 for case iscommunicated with the through-hole 1314-1 for auxiliary covering partand the provisional storing part 1312, respectively.

Referring to FIGS. 19 and 21, a case cover 1500 is attached to thecoupling case 1400. The case cover 1500 is formed with a through-hole1524 for case cover which passes through upper and lower surfacesthereof. The through-hole 1524 for case cover is formed so that thethrough-hole 1314-1 for auxiliary covering part is exposed to an outsideand the provisional storing part 1312 is partially closed, when the casecover 1500 is attached to the coupling case 1400. A reference numeral1500S in FIG. 19 is a case cover set in which multiple case covers 1500are connected with each other.

Other elements which are not described are based on the description ofthe fifth embodiment.

According to the present invention as described above, since theseparation membrane which is the injection part of the sample solutionincluding nucleic acid is used as the optical measuring part, it ispossible to provide a simple structure, prevent measurement error of theoptical measuring part due to the contamination, reduce a size of theanalytic micro-chamber plate, facilely control the temperature and thusremarkably reduce the analyzing time.

Further, in case that the sample solution including nucleic acid isinjected into the chamber hole, since the gas in the chamber hole isfirstly removed by using vacuum, and then the injection of the samplesolution is performed through the separation membrane, it is possible tocompletely inject the sample solution within a short time without anyremained gas and prevent the error of the optically measured value dueto the remain gas. Further since the separation membrane is sealed withthe polymer oil such as mineral oil and silicon oil, it is possible toprevent the cross contamination due to the mixing of the solutions inthe chamber holes, thereby increasing the analyzing accuracy.

Further, since the multiple analytic micro-chamber plates can be formedintegrally, it is possible to compare and analyze various kinds ofsamples at the same time, thereby remarkably reducing the analyzingtime.

Further, since the separation membrane and the other surface of theoptical measuring part can be integrally formed with the analyticmicro-chamber plate, the analytic micro-chamber plate of the presentinvention can be manufactured by the compression molding of aluminum orthe like, and thus the production process and manufacturing cost can beremarkably reduced.

While the present invention has been described with respect to thespecific embodiments, it will be apparent to those skilled in the artthat various changes and modifications may be made without departingfrom the spirit and scope of the invention as defined in the followingclaims.

What is claimed is:
 1. A method of manufacturing a micro-chamber platewith a built-in sample, comprising: settling a micro-chamber plate forsample injection at a micro-chamber plate receiving part formed with anupper opening; disposing a cover on the micro-chamber plate receivingpart so as to cover the upper opening of the micro-chamber platereceiving part, the cover comprising: a provisional storing part formedwith a cutting line; and an auxiliary covering part connected with theprovisional storing part and formed with a through-hole; putting themicro-chamber plate receiving part, on which the cover is disposed, intoa centrifugal separator which is capable of applying centrifugal forceand vacuum into the centrifugal separator; and injecting a samplesolution provisionally stored in the provisional storing part into themicro-chamber plate for sample injection through the cutting line whichis formed at the provisional storing part so as to be communicated withthe micro-chamber plate receiving part.
 2. The method of claim 1,wherein the injecting a sample solution provisionally stored in theprovisional storing part into the micro-chamber plate comprises:applying the vacuum into the centrifugal separator and generating thecentrifugal force at a first level while the vacuum is applied into thecentrifugal separator; and releasing the vacuum in the centrifugalseparator while generating the centrifugal force at a second levellarger than the first level in the centrifugal separator such that thesample solution is injected into the micro-chamber plate through thecutting line.
 3. The method of claim 2, wherein the centrifugal force atthe first level is configured to suppress bumping of the samplesolution, while the vacuum is applied into the centrifugal separator andthe cutting line is closed.
 4. The method of claim 3, wherein thecutting line is formed at the provisional storing part so as to beopened by external force, and the centrifugal force at the second levelis configured to open the cutting line.
 5. A method of manufacturing ananalytic micro-chamber plate using the micro-chamber plate with thebuilt-in sample manufactured by the method of claim 1, comprising:taking out the micro-chamber plate with the built-in sample from thecentrifugal separator and then sealing a separation membrane of themicro-chamber plate with the built-in sample.
 6. A method ofmanufacturing a micro-chamber plate with a built-in sample, comprising:forming a sample solution storing space between a cover for amicro-chamber plate receiving part and an upper surface of amicro-chamber plate for sample injection by closely contacting a lowerend of the cover to the upper surface of the micro-chamber plate forsample injection, the cover comprising: a provisional storing partformed with a cutting line; and an auxiliary covering part connectedwith the provisional storing part and formed with a through-hole;putting the micro-chamber plate for sample injection and the cover,which are closely contacted with each other so as to form the samplesolution space therebetween, into a centrifugal separator which iscapable of applying centrifugal force and vacuum into the centrifugalseparator; and injecting a sample solution provisionally stored in theprovisional storing part into the micro-chamber plate for sampleinjection through the cutting line which is formed at the provisionalstoring part so as to be communicated with the sample solution storingspace.
 7. The method of claim 6, wherein the injecting a sample solutionprovisionally stored in the provisional storing part into themicro-chamber plate comprises: applying the vacuum into the centrifugalseparator and generating the centrifugal force at a first level whilethe vacuum is applied into the centrifugal separator; and releasing thevacuum in the centrifugal separator while generating the centrifugalforce at a second level larger than the first level in the centrifugalseparator such that the sample solution is injected into themicro-chamber plate through the cutting line.
 8. The method of claim 7,wherein the centrifugal force at the first level is configured tosuppress bumping of the sample solution, while the vacuum is appliedinto the centrifugal separator and the cutting line is closed.
 9. Themethod of claim 8, wherein the cutting line is formed at the provisionalstoring part so as to be opened by external force, and the centrifugalforce at the second level is configured to open the cutting line.
 10. Amethod of manufacturing an analytic micro-chamber plate using themicro-chamber plate with the built-in sample manufactured by the methodof claim 6, comprising: taking out the micro-chamber plate with thebuilt-in sample from the centrifugal separator and sealing a separationmembrane of the micro-chamber plate with the built-in sample.